Targeted Sanger sequencing to recover key mutations in SARS-CoV-2 variant genome assemblies produced by next-generation sequencing.
Targeted Sanger sequencing to recover key mutations in SARS-CoV-2 variant genome assemblies produced by next-generation sequencing.
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DOI：
10.1099/mgen.0.000774
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发表时间：
2022-03
影响因子：
3.9
通讯作者：
de Oliveira, Tulio
中科院分区：
文献类型：
--
作者：
Singh, Lavanya;San, James E.;Tegally, Houriiyah;Brzoska, Pius M.;Anyaneji, Ugochukwu J.;Wilkinson, Eduan;Clark, Lindsay;Giandhari, Jennifer;Pillay, Sureshnee;Lessells, Richard J.;Martin, Darren Patrick;Furtado, Manohar;Kiran, Anmol M.;de Oliveira, Tulio
关键词：
严重急性呼吸综合征冠状病毒2(SARS-CoV-2)是适应性进化的，以确保其在人类宿主中的持久性。因此，有必要持续监测新变种的出现和流行情况。重要的是，一些突变既与分子诊断失败有关，也与某些基因组区域下一代测序(NGS)读数覆盖率降低或取消有关。当这种影响发生在基因组区域时尤其有问题，例如编码尖峰蛋白(S)的那些区域，这些区域对于识别和跟踪相关病毒变异的流行和传播动态至关重要。靶向Sanger测序提供了一种快速和经济有效的方法来准确地扩大全基因组序列的覆盖范围。我们设计了一套定制的引物来扩增受体结合结构域(相对于武汉-HU-1参考文献的22698至23098位之间)的401bp片段。然后，我们设计了桑格测序的湿实验室方案。我们对222个因NGS测序覆盖率较低而缺失关键突变K417N、E484K和N501Y的样本进行了测序。最后，我们开发了SeqPatcher，这是一个基于Python的计算工具，用于分析Sanger测序产生的跟踪文件，以生成一致序列，或以FASTA格式获取预先分析的一致序列，并将它们与相应的全基因组组合合并。对2 2 2例 样本中的15 3例(69 %)进行了测序，证实了其中14 2例(93 %)S基因存在关键的β突变(K417N、E484K、N501Y)。此外，1个样本有Y508F突变，4个 样本有S477N突变。RT-PCRCt评分在13.85~37.47之间(平均值为25.70)的样本可以有效地进行Sanger测序。这些结果表明，我们的方法和流水线可以用来提高使用NGS生产的全基因组组件的质量，并且可以用于任何一对最常用的NGS和Sanger测序平台。
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is adaptively evolving to ensure its persistence within human hosts. It is therefore necessary to continuously monitor the emergence and prevalence of novel variants that arise. Importantly, some mutations have been associated with both molecular diagnostic failures and reduced or abrogated next-generation sequencing (NGS) read coverage in some genomic regions. Such impacts are particularly problematic when they occur in genomic regions such as those that encode the spike (S) protein, which are crucial for identifying and tracking the prevalence and dissemination dynamics of concerning viral variants. Targeted Sanger sequencing presents a fast and cost-effective means to accurately extend the coverage of whole-genome sequences. We designed a custom set of primers to amplify a 401 bp segment of the receptor-binding domain (RBD) (between positions 22698 and 23098 relative to the Wuhan-Hu-1 reference). We then designed a Sanger sequencing wet-laboratory protocol. We applied the primer set and wet-laboratory protocol to sequence 222 samples that were missing positions with key mutations K417N, E484K, and N501Y due to poor coverage after NGS sequencing. Finally, we developed SeqPatcher, a Python-based computational tool to analyse the trace files yielded by Sanger sequencing to generate consensus sequences, or take preanalysed consensus sequences in fasta format, and merge them with their corresponding whole-genome assemblies. We successfully sequenced 153 samples of 222 (69 %) using Sanger sequencing and confirmed the occurrence of key beta variant mutations (K417N, E484K, N501Y) in the S genes of 142 of 153 (93 %) samples. Additionally, one sample had the Y508F mutation and four samples the S477N. Samples with RT-PCR C t scores ranging from 13.85 to 37.47 (mean=25.70) could be Sanger sequenced efficiently. These results show that our method and pipeline can be used to improve the quality of whole-genome assemblies produced using NGS and can be used with any pairs of the most used NGS and Sanger sequencing platforms.
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期刊：
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DOI：
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